Membrane proteins are of critical importance to nearly every aspect of cell physiology, comprising quarter to one third of all proteins encoded by eubacterial, archaean, and eukaryotic organisms. Nevertheless, our understanding of their folding and structures is at a very primitive state when compared to that of water-soluble proteins. The M2 proton channel from influenza A virus provides an attractive system for understanding the folding and function of the viroporins. This growing class of helical viral ion channels is important for viral replication and packaging, and also provides good pharmaceutical targets. The M2 proton channel is essential to the survival of the virus, and is the target of the anti-influenza drugs, amantadine and rimantadine. The development of resistant strains of the virus has limited the effectiveness of these drugs, highlighting the importance of determining the structure of the protein-drug complex. The M2 proton channel is formed by the intermolecular association of four identical trans-membrane helices, and a synthetic peptide (M2tm) spanning only the trans-membrane helix of M2 forms amantadine-dependent channels in bilayers. Our specific aims for the current period are to: 1. Determine the contributions of specific side-chains and the bilayer composition to the folding of M2tm and the full-length protein. 2. Using synthetic peptides and recombinant proteins we will examine the role of specific side-chains and structural features in the mechanism of channel gating, conductance, and inhibition by conducting ion channel measurements in vesicles and planar bilayers. In particular, we will evaluate the role of His37, which is essential for both gating and conductance, and its interactions with Trp41. 3. Use spectroscopic approaches (oriented CD, time-resolved fluorescence, NMR, EPR) to probe how changes in lipid composition, pH, and inhibitor binding affect the overall quaternary structure of the channel as well as the dynamics of side-chains involved in the function of the protein. 4. Determine the NMR structure of the full-length protein reconstituted in bicelles at pH 5, 8, and in the amantadine inhibited complex. 5. Determine the crystal structure of M2tm at pH 5, 8, and in the amantadine-inhibited complex.